FEMS Microbiology Ecology 62 (1989) 71-78 71 Published by Elsevier

FEC 00192

Field determination of denitrification in water-logged forest soils

Sten S t ruwe a n d Anne l i se Kjo l l e r

Department of General Microbiology, University of Copenhagen, Copenhagen, Denmark

Received 1 April 1988 Revision received 27 June 1988 Revision accepted 28 June 1988

Key words: Rate of denitrification; Acetylene blockage technique; Soils, alder and ash; N20 production; Field method

1. S U M M A R Y 2. I N T R O D U C T I O N

Denitrification rates were measured as N20 production in two water-logged forest soils at monthly intervals. The effect of acetylene inhibi- tion and the addition of nitrate, glucose, acetate and cellobiose in field incubations was examined.

N20 release from the two soils was very low, 26 mg NEm-Ey -1 in ash and 178 mg N 2 in alder. In acetylene inhibited incubations N20 production was higher, 296 and 486 mg N2 m - 2 y-1 in ash and alder respectively. After addition of nitrate and C-sources to a 10 m M concentration, denitri- fication rates increased to 5-15 times higher val- ues.

The denitrification rates below 4 ° C were low and most N20 was produced in late spring and summer.

The highest rate of denitrification during a 50 h incubation experiment occurred between 3 and 23 h.

Correspondence to: S. Struwe, Department of General Microbi- ology, University of Copenhagen, Solvgade 83H, DK-1353 Copenhagen K, Denmark.

Denitrification has traditionally been consid- ered a sink in the construction of nitrogen budgets in ecosystems. More detailed knowledge of the process was only acquired after two major innova- tions. Gas chromatography was introduced in field work by Payne [1], and combined with the dis- covery of Fedorova, Milekhina and I I 'Yukhina [2], that acetylene inhibited nitrous oxide re- ductase, a new and easy method for direct mea- surement of denitrification was available [3].

In situ measurements of denitrification have been carried out in soil or sediment cores brought to the laboratory [4-9]. Other researchers have established chambers in the field, equipped with a continuous air-flow through the non-subterranean part of the chamber, and N20 production was measured [10,11]. A recent paper [27] recommends incubation of soil cores to obtain fast distribution of C2H2, when poorly drained soils are examined. In reports on denitrifying activity in slurries from marine sediments, a distinct seasonal variation has been shown and a temperature selection in the population of denitrifiers demonstrated [23,24]. Jacobsen and Alexander [16] examined the de- nitrifying activity of isolates of denitrifiers grown

0168-6496/89/$03.50 © 1989 Federation of European Microbiological Societies

72

exponentially in pure cultures. The denitrifying capacity was compared with denitrification rates obtained in soil samples. All but one of ten soil samples showed higher denitrification rates per cell than the pure cultures, giving support to the idea of experiments under natural conditions.

In this paper we present a field method for collection of N20 produced in water-logged forest soils. The advantage of the method is that it causes very little disturbance of the system during the in situ measurements. The method was tested to determine the rate and magnitude of denitrifi- cation, and to examine the effect of acetylene inhibition in the field. The potential denitrifica- tion in situ was measured simultaneously after addition of nitrate and carbon sources: acetate, glucose or cellobiose. The measurements were car- ried out during most of the frost-free months for one year f rom September 1984 until August 1985, to demonstrate any seasonal variation. In previous studies monthly determinations of the numbers of denitrifiers in the two stands were carried out, using both the MPN method and the roll-tube technique [12,13].

The investigated sites were stands of alder (AI- nus glutinosa) and ash ( Fraxinus excelsior) de- scribed in Kjoller and Struwe [14], Struwe and Kjoller [13], and Westermann and Ahring [15,26]. The soils were water-logged during the summer period and partially flooded in the winter. The surface of the soils was frozen in January, February and March. The soil temperature rose from 0 ° C in March to 1 4 - 1 6 ° C in July, and fell to 4 ° C in November.

The soil pH values in the alder and ash stands varied between 6.5-7.0 and 6.5-7.5, respectively, measured in a slurry of wet soil and deionized water 1 : 1.

The total nitrogen content in alder litter was high, partially as a result of the nitrogen-fixing symbiosis between alder and the micro-symbiont Frankia, with an initial nitrogen content of 2.1% increasing to 2.6% at the end of the decomposition period, in June [12]. The nitrogen content in the ash-litter was also very high despite the absence of a nitrogen-fixing symbiosis and the nitrogen con- tent increased from 1.5 to 3.0% during decomposi- tion. The litter layer of each stand decreased from

approx. 3 cm to less than 1 cm during decomposi- tion [13].

3. M A T E R I A L A N D M E T H O D S

One liter serum bottles equipped with rubber septa and with bot toms cut off were inserted into the water-logged soil to the 500 ml mark. Ad- ditions of potassium nitrate and carbon sources (acetate, glucose or cellobiose) were made as injec- tions of 10 ml 0.5 M solution of each substrate through the septum at the initiation of each ex- periment. The additions were distributed on the surface of the enclosed area, but the distribution with depth was not measured. The resulting con- centration in the 0.5 1 volume enclosed by the bottles would amount to 10 mM, corresponding to 13 m M in the water phase.

Different substrate combinations were tested in duplicate incubations and the in situ incubation period was 24 h. A 50 h incubation experiment with the same additions was carried out in the alder stand in October. N20 was measured after 3, 12, 23, and 50 h incubation. A 24 h incubation experiment was carried out in November in the alder stand comparing the rate of denitrification using different combinations of nitrate and acetate concentrations (1, 5, 10 and 20 mM of one com- pound and 10 mM of the other).

Acetylene blockage of the reduction of N20 to N 2 was used to stop denitrification at the N20 step with 10% acetylene in the headspace of the serum bottles. The acetylene was generated from calcium carbide in the field. Gas samples (1 ml) were drawn with gas-tight syringes and trans- ported to the laboratory within 1 h and the N20 concentration was determined on a gas chromato- graph (Packard 427) equipped with an electron capture detector on a 3 m stainless steel column packed with Porapak Q. The injector, oven and detector temperatures were 100, 60 and 320 ° C, respectively. Purified nitrogen was used as carrier gas, the flowrate was 15 ml min -1. All samples were analyzed in duplicate.

No corrections have been made for the amount of N20 solubilized in the water phase [25] of the upper 2 cm of the soil, where the major part of the

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1 5 10 210 mM NO~//acetate Fig. 3. Denitrifi_cation rate as a function of different combina- tions of acetate and nitrate additions m- field incubations at 10 o C. In experiments with four different acetate or nitrate concentrations, nitrate or acetate respectively, was added.

den i t r i f i ca t ion takes p lace [15]. The re la t ive ly long incuba t ion pe r iod of 24 h was used to ensure m a x i ma l accumula t ion of N 2 0 in the 500 ml head space, as shown in the 50 h exper iment .

4. R E S U L T S

The results of this work d e m o n s t r a t e d i rec t ly the seasonal change in deni t r i f ica t ion. Al l i ncuba - t ions w e r e m a d e in the f ield whe ther u n a m e n d e d or with a d d e d n i t r a t e and ca rbon source. The measurement s in the a lder s t and were a lways car- r ied out the week preced ing the exper imen t in the ash s tand. The soil t empera tu re was usua l ly h ighest in the a lder s tand.

The measured da i ly rates of den i t r i f i ca t ion are p resen ted in Figs. 1 and 2. The act iv i ty was uni - fo rmly high f rom M a y to August . In Apr i l , Oc- tober , N o v e m b e r and D e c e m b e r there were on ly

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Table 2

The estimated, hourly denitrification rate in the alder stand in October at 10 o C, expressed as rng N 2 m-2 h-1

Additions 0-3 h 3-12 h 12-23 h 23-50 h

NO 3 0.13 0.17 0.11 0.15 N O r , C2H 2 0.18 0.15 0.11 0.14 Glucose + + 0.18 0.33 0.32 0.19 Acetate + + 0.07 0.24 0.25 0.16 Cellobiose + + 0.20 0.29 0.30 0.21

+ + = NO~- and C2H 2

lOW values and these con t r i bu t ed very l i t t le to the to ta l annua l den i t r i f i ca t ion . In Sep t ember the ac- t iv i ty level was i n t e rme d ia t e be tween the summer and la te a u t u m n level.

The es t imates of den i t r i f i ca t ion rates for all m e a s u r e m e n t s shown (9 mon ths for a lder and 6 mon ths for ash) were used in ca lcu la t ing the annua l N 2 p r o d u c t i o n shown in Tab le 1. The ra te of d e n i t r i f i c a t i o n as measu red b y N 2 0 p roduc t i on was very low in b o t h sys tems when unamended , whe ther inh ib i t ed wi th ace ty lene or not. The highest values were seen in the a lder s tand. The a d d i t i o n of n i t r a te inc reased the annua l deni t r i f i - ca t ion ra te in the ash s t and to a s l ightly h igher level than in the a lder system. A d d i t i o n of bo th n i t ra te and aceta te , g lucose or ce l lobiose showed stil l h igher values. The highest annua l p roduc t ion was f o u n d in the a lder sys tem (Tab le 1), while the h ighest va lue in an ind iv idua l mon th , July, was r eco rded in the ash s t and (Figs. 1 and 2).

The hou r ly ra te o f den i t r i f i ca t ion dur ing the 50 h expe r imen t in the a lder s t and is shown in Tab le 2. The highest ra tes o f den i t r i f i ca t ion were found af ter 3 - 1 2 a n d 1 2 - 2 3 h of i ncuba t ion in the C- and n i t r a t e - a m e n d e d exper iments .

The expe r imen t wi th d i f ferent concen t ra t ions o f n i t r a te and ace ta te c o n d u c t e d in the a lder s t and

Table 1

The estimated, annual denitrification rate in the alder and ash stands expressed as mg N 2 m - 2 y-1

No additions C 2 H 2 NOj- NOj- Acetate C2H2 + +

Aider 177.9 486.0 1617 2595 4209 Ash 26.4 295.5 2366 2640 3622

+ + = NOr and C2H 2

Glucose Cellobiose + + + +

4653 4962 4060 4614

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(Fig. 3), shows optimal conditions for denitrifica- tion with acetate added to a 5 mM concentration. The denitrification rates were equally high with 5 and 10 mM nitrate.

5. DISCUSSION

All measurements of denitrification were based on N20 production and the effect of acetylene inhibition. The inhibitory effect of acetylene on N 2 0 reduction in the soil system can be found from the figures in Table 1, comparing treatments with and without C2H 2. In the unamended sys- tems NEO-production was 2.7-11 times higher with acetylene than without and in the nitrate amended system only 1.1-1.6 times higher. The effect of acetylene was also examined in the incubations with cellobiose and nitrate at two consecutive samplings in June and July and the N 2 0 concentration was 1.1-1.2 times higher with acetylene than without (data not shown). This is in agreement with the observation of Betlach and Tiedje [22] that at high nitrate and substrate con- centrations, nitrate reduction does not proceed further than the nitrous oxide level, and N 2 0 is the end-product. It is concluded that acetylene exhibits its inhibitory effect in this wet soil system, but is most pronounced in the non-amended incubations. It has not been shown how deep acetylene penetrates, but most of the denitrifying activity has been found in the upper 2 cm [15].

The seasonal variation in activity (Figs. 1 and 2) responded to the seasonal change in tempera- ture, while the number of denitrifying bacteria enumerated during previous years showed less sea- sonal variation [12,13]. In the period with the most intensive denitrification, the denitrifying popu- lation (roll-tube counts) was shown to consist of approx. 106 bacteria ga of dry surface soft in the ash stand, and approx. 5 × 106 bacteria in the alder stand. At 3 - 4 ° C the denitrifying activity was very low, virtually no denitrification occurred in the unamended soil (with or without acetylene) and denitrification was also low in the correspond- ing nitrate and substrate enriched plots.

When incubated with addition of nitrate (10 mM conc.) the maximal figures for denitrification

were found in May, 20 and 28 mg N 2 m - 2 d-1 in the alder and ash stands, respectively. The corre- sponding figures for the cold months were 2-5 mg N 2 m -2 d -1. After an increase of the rate of denitrification in May the activity reached a high summer level maintained in the amended plots until September. Addition of carbon sources to a 10 mM concentration further increased the annual denitrification 37-91%. The response to the ad- ditions of nitrate and carbon sources showed that the two systems were both N- and C-limited.

The temperature effect in field incubations has recently been examined by Lensi and Chalamet [17] and a highly temperature-dependent response was seen. In the morning incubations at 10 ° C, they found lower hourly denitrification rates than in our 50 h experiment (Table 2) but during the day the rate increased ten times simultaneous with an increase in temperature from 10-20°C. The soil temperature remained constant (10 o C) during our 50 h experiment, and the hourly N20 produc- tion only varied from 0.11-0.18 mg N 2 m -2. Few other field denitrification experiments have been reported from wet or flooded soils, and only a few comparable values are available. In situ measure- ments in freshwater sediments ranged from 5-96 mg N 2 m -2 d -1 [18] and a flooded field showed a flux of 30 mg N 2 m -2 d -1 [19].

In a recent investigation in the Florida Everg- lades, Gordon et al. [20] determined the denitrifi- cation rate in laboratory incubations of soil sam- ples. One of the soil types, comparable with our organic soils, was a peat with a denitrification rate in the winter ranging from 1-5 nmol N 2 100 m1-1 min-1 after addition of nitrate. Our field incuba- tions with addition of nitrate and inhibited with acetylene evolved (converted to molarity and ml) 0.2-2.5 nmol N 2 100 m1-1 rain -1 in the surface soil varying during the year. The denitrifying ac- tivity in the Everglades peat soil did not increase upon addition of glucose in contrast to our ash and alder soils where denitrification only reached 5 nmol N 2 100 m1-1 rain -1 after addition of carbon sources.

The denitrification rates found in the two forest stands were low, as in most natural ecosystems. If other end products accumulated or the N20 blockage was not complete, the total denitrifica-

t ion would be larger than est imated f rom N20. The month ly sampl ing p rogramme could overlook t rans ient activity peaks, bu t they are no t likely to occur in the un i fo rm and water-logged system. Despite some difference in the individual mea- surements no indica t ion of the existence of hot spots with high activity was seen [21].

Seasonal changes in a denitr if ier c o m m u n i t y f rom a salt marsh were investigated by K ing and Nedwell [24], who throughout the year found mesophil ic bacteria, bu t only dur ing the winter a psychrotrophic popula t ion . The popula t ions were described by the frequency of genera isolated from enr ichment cultures. At 1 0 ° C Pseudomonas iso- lates accounted for 98% of the isolates and at

25 ° C 94% of the isolates belonged to the Vibrio

genus. Our pre l iminary results f rom the ash and alder swamps showed correspondingly a domina t - ing Pseudomonas popula t ion in the spring.

The field incuba t ion method provides less favorable condi t ions for deni t r i f icat ion than a cor- responding slurry experiment. Especially diffusion between surface and deeper soil layers and gas exchange could be l imiting. In spite of these disad- vantages we have chosen to conduct our experi- ments as in situ incuba t ions to s imulate na tu ra l and und i s tu rbed condi t ions. This makes compari- sons with the impor t an t a m o u n t of work on slurry incuba t ions in the labora tory difficult. In subse- quen t work s imul taneous de te rmina t ion of the activity in the field and in slurry incuba t ions will be carried out.

R E F E R E N C E S

[1] Payne, W.J. (1973) The use of gas chromatography for studies of denitrification in ecosystems. BuU. Ecol. Res. Comm. (Stockholm) 17, 263-268.

[2] Fedorova, R.I., Milekhina, E.I. and II'Yukl'6na, N.I. (1973) Evaluation of the method of 'gas metabolism' for detecting extraterrestrial life. Izv. Akad. Nauk. SSSR Ser. Biol. 6, 797-806.

[3] Balderston, W.L., Sherr, B. and Payne, W.J. (1976) Bloc- kage by acetylene of nitrous oxide reduction in Pseudo- monas perfectomarinus. Appl. Environ. Microbiol. 31, 504-508.

[4] Sorensen, J. (1978) Capacity for denitrification and reduc- tion of nitrate to ammonia in coastal marine sediment. Appl. Environ. Microbiol. 35, 301-305.

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[5] Christensen, S. (1980) Percolation studies on denitrifica- tion. Acta Agricul. Scand. 30, 225-236.

[6] Nishio, T., Koike, I. and Hattori, A. (1982) Denitrifica- tion, nitrate reduction and oxygen consumption in coastal and estuarine sediments. Appl. Environ. Microbiol. 43, 648-653.

[7] Parkin, T.B. and Tiedje, J.M. (1984) Application of a soil core method to investigate the effect of oxygen concentra- tion on denitrification. Soil Biol. Biochem. 16, 331-334.

[8] Parldn, T.B., Kaspar, H.F., Sextone, A.J. and Tiedje, J.M. (1984) A gas flow soil core method to measure field denitrification rates. Soil Biol. Biochem. 16, 323-330.

[9] Christensen, P.B., and Sorensen, J., (1986) Temporal vari- ation of denitrification activity in plant-covered, litoral sediment from Lake Hampen, Denmark. Appl. Environ. Microbiol. 51, 1174-1179.

[10] Chan, Y-K., and Knowles, R. (1979) Measurement of denitrification in two freshwater sediments by an in situ acetylene inhibition method. Appl. Environ. Microbiol. 37, 1067-1072.

[11] Christensen, S. (1983) Nitrous oxide emission from a soil under permanent grass: Seasonal and diurnal fluctuations as influenced by manuring and fertilization. Soil Biol. Biochem. 15, 531-536.

[12] Kjoller, A., Struwe, S. and Vestberg, K. (1985) Bacterial dynamics during decomposition of alder litter. Soil Biol. Biochem. 17, 463-468.

[13] Struwe, S. and Kjoller, A. (1985) Functional groups of bacteria on decomposing asia litter. Pedobiol. 28, 367-376.

[14] Kjoller, A. and Struwe, S. (1980) Microfungi in decompos- ing red alder and their substrate utilization. Soil. Biol. Biochem. 12, 425-431.

[15] Westermann, P. and Ahring, B.K. (1986) Terminal anaerobic carbon mineralization in an alder swamp. FEMS Symposium 33, in Microbial Communities in Soil (V. Jensen, A. Kjoller and L.H. Sorensen, eds.), pp. 305-315. Elsevier Applied Science Publishers, London and New York.

[16] Jacobsen, S.N. and Alexander, M. (1980) Nitrate loss from soil in relation to temperature, carbon source and denitrifier populations. Soil Biol. Biochem. 12, 501-505.

[17] Lensi, R. and Chalamet, A. (1982) Denitrification in waterlogged soils: In situ temperature-dependent varia- tions. Soil Biol. Biochem. 14, 51-55.

[18] Tiren, T. (1977) Denitrification in sediment and water systems of various types of lakes, in Interactions between sediment and fresh water. (H.L. Goltermann, ed.), pp. 363-369. W. Jung Publishers, The Hague.

[19] Denmead, O.T., Freney, J.R. and Simpson, J.R. (1979) Nitrous oxide emission during denitrification in a flooded field. Soil Sci. Soc. Am. J. 43, 716-718.

[20] Gordon, A.S., Cooper, W.J. and Scheit, D.J. (1986) De- nitrification in marl and peat sediments in the Florida Everglades. Appl. Environ. Microbiol. 52, 987-991.

[21] Groffman, P.M., Christensen, S., Rice, C.W., and Tiedje, J.M. (1986) Understanding the causes of spatial and tem- poral variability of denitrification. Abstracts. Fourth In-

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ternational Symposium on Microbial Ecology. ICOME Conference. Ljubljana, Yugoslavia. August 24-29, 1986.

[22] Betlach, M.R. and Tiedje, J.M. (1981) Kinetic explanation for accumulation of nitrite, nitric oxide and nitrous oxide during bacterial denitrification. Appl. Environ. Microbiol. 42, 1074-1084.

[23] Kaplan, W.A., Teal, J.M. and Vallela, I. (1977) Denitrifi- cation in salt marsh sediments: evidence for seasonal temperature selection among population of denitrifiers. Microbiol. Ecol. 3, 193-204.

[24] King, D. and NedweU, D.B. (1984) Changes in the nitrate-reducing community of an anaerobic saltmarsh

sediment in response to seasonal selection by temperature. J. Gen. Microbiol. 130, 2935-2941.

[25] Weiss, R.F. and Price, B.A. (1980) Nitrous oxide solubility in water and seawater. Mar. Chem. 8, 347-359.

[26] Westermann, P. and Ahring, B.K. (1987) Dynamics of methane production, sulfate reduction, and denitrification in a permanently waterlogged alder swamp. Appl. En- viron. Microbiol. 53, 2554-2559.

[27] Ryden, J.C., Skinner, J.H. and Nixon, D.J. (1987) Soil core incubation system for the field measurement of de- nitrification using acetylene inhibition. Soil Biol. Biochem. 19, 753-757.